Biotinylation Of Cell Surface Proteins Research Articles

Influence of glycoprotein MUC1 on trafficking of the Ca2+-selective ion channels, TRPV5 and TRPV6, and on in vivo calcium homeostasis

Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5, in cultured unpolarized cells. Using biotinylation of cell surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.

Identification of Novel Antigens for Normal Karyotype Triple Mutated Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. AML is divided in several subgroups based on genetic alterations and prognosis varies among AML subgroups. Normal karyotype AML with NPM1, DNMT3A and FLT3(ITD) mutations (NK triple mutated AML) is associated with a long-term overall survival of ~20% and represents ~6% of AML cases (Papaemmanuil et al., NEJM, 2016). No curative chemotherapy for this subgroup of AML patients currently exists, highlighting the need to develop novel therapeutic strategies for this group of AML patients. Methods To identify novel cell surface antigens for NK triple mutated AML that could ultimately be targeted by immunotherapeutic approaches, we developed a method to map the surface proteome of primary human AML specimens based on biotinylation of cell surface proteins, affinity purification and mass spectrometry. To further enrich mass spectrometry datasets with surface proteins, we developed an algorithm that assesses the probability of a protein to be located on the cell surface based on annotations from various databases. Using this approach, the surface proteome of 100 primary human AML specimens was analysed, which included 12 NK triple mutated AML samples. Differential surfaceome analyses identified several surface proteins specifically expressed by NK triple mutated AML specimens. Expression analyses were performed using publicly available datasets to select proteins with limited expression in normal tissues and hematopoietic cells. Surface expression and AML subgroup specificity for these potential antigens was evaluated by flow cytometry using primary human AML specimens from various AML subgroups (n=76). Moreover, to assess the intra-specimen expression profile of these antigens, single cell RNA-sequencing of 15 primary human NK triple mutated AML specimens was performed. Finally, we evaluated the prognostic impact of antigen expression on overall survival in a subgroup of patients from the Leucegene cohort composed of patients diagnosed with de novo AML with intermediate risk cytogenetics who have been treated with intensive induction chemotherapy (n = 316). Results Surface proteome analysis of NK triple mutated AML specimens and hit filtering according to selection pipeline described above identified several promising NK triple mutated AML antigens. Surface expression analysis of these antigens by flow cytometry showed specificity of antigen expression for NK triple mutated AML specimens. Transcriptomic analyses using RNA sequencing data from primary human AML specimens of the Leucegene cohort (n=691) showed significantly higher antigen transcript levels in NK triple mutated AML specimens compared to samples from other AML subgroups. Moreover, multivariate transcriptome analysis comparing the impact of most frequent mutations in AML on antigen expression revealed that FLT3-ITD mutation is the only significant factor associated with antigen overexpression in AML samples. Single cell RNA sequencing data from NK triple mutated AML specimens revealed that these antigens are uniformly expressed across the vast majority of blast populations, including blast cells with an expression profile reminiscent of leukemic stem cells. Interestingly, in patients with intermediate cytogenetic risk AML, high antigen expression was significantly associated with worse overall survival. Patients with high antigen expression who proceeded to allogeneic hematopoietic stem cell transplantation (HSCT) in first complete remission also had a significantly lower 5-year overall survival rate post-HSCT compared to patients with low antigen expression. Conclusion We identified novel antigens for FLT3-ITD mutated and NK triple mutated AML, which show homogeneous expression in blast populations of AML samples including most primitive ones, making it ideal antigens for immunotherapeutic targeting. Patients with intermediate cytogenetic risk AML and high antigen expression show poor prognosis even after HSCT in first complete remission, highlighting the need to develop novel therapeutic strategies for these patients. Different immunotherapeutic approaches targeting these antigens are currently being developed by our group, with the hope of identifying novel therapeutic strategies for the treatment of NK triple mutated AML.

Cell surface biotinylation to identify the receptors involved in nanoparticle uptake into endothelial cells

Targeted drug delivery requires –among others- specific interaction of nanocarriers with cell surface receptors enabling efficient internalization into the targeted cells. Thus, identification of receptors allowing efficient nanocarrier uptake is essential to improve the design of targeted nanomedicines. Here we used methods based on cell surface biotinylation to identify cell surface receptors mediating nanoparticle uptake by cells. We used human brain and liver endothelial cells as representative examples of cells typically showing very low and very high nanoparticle uptake, respectively. Amino-modified and carboxylated silica were used as model nanoparticles usually associated with high and low uptake into cells, respectively, and carrying different coronas after exposure in full human plasma. Using cell surface biotinylation of live cells and receptor pull-down assays, we compared the receptors internalized in control untreated cells and those internalized upon exposure to nanoparticles. In this way, we identified receptors associated with (high) nanoparticle uptake. The candidate receptors were further validated by decorating the nanoparticles with an artificial corona consisting of the respective receptor ligands. We found that a vitronectin corona can be used to target integrin receptors and strongly enhances nanoparticle uptake in brain and liver endothelial cells. The increased uptake was maintained in the presence of serum, suggesting that the vitronectin-corona could resist interaction and competition with serum. Furthermore, plasminogen-coated nanoparticles promoted uptake in endothelial cells of the liver, but not of the brain. The presented approach using reversible biotinylation of cell surface receptors in live cells allows for receptor-based targeting of nanocarriers that are instrumental in nanoparticle uptake, which can be exploited for targeted drug delivery. Statement of significanceIn order to deliver drugs to their site of action, drug-loaded nanocarriers can be targeted to cell receptors enabling efficient uptake into target cells. Thus, methods to identify nanocarrier receptors are invaluable. Here we used reversible biotinylation of live cells and receptor pull-down approaches for receptor identification. By comparative analysis of the individual receptors internalized in untreated cells and cells exposed to nanoparticles, we identified receptors enabling high nanoparticle uptake into liver and brain endothelial cells. Their role was confirmed by decorating nanoparticles with an artificial corona composed of the receptor ligands. In conclusion, live cell reversible biotinylation of cell surface proteins is a powerful tool for the identification of potential receptors for receptor-based targeting of nanocarriers.

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.

Surfaceome Proteomic of Glioblastoma Revealed Potential Targets for Immunotherapy.

Glioblastoma (GBM) is the most common and devastating malignant brain tumor in adults. The mortality rate is very high despite different treatments. New therapeutic targets are therefore highly needed. Cell-surface proteins represent attractive targets due to their accessibility, their involvement in essential signaling pathways, and their dysregulated expression in cancer. Moreover, they are potential targets for CAR-based immunotherapy or mRNA vaccine strategies. In this context, we investigated the GBM-associated surfaceome by comparing it to astrocytes cell line surfaceome to identify new specific targets for GBM. For this purpose, biotinylation of cell surface proteins has been carried out in GBM and astrocytes cell lines. Biotinylated proteins were purified on streptavidin beads and analyzed by shotgun proteomics. Cell surface proteins were identified with Cell Surface Proteins Atlas (CSPA) and Gene Ontology enrichment. Among all the surface proteins identified in the different cell lines we have confirmed the expression of 66 of these in patient’s glioblastoma using spatial proteomic guided by MALDI-mass spectrometry. Moreover, 87 surface proteins overexpressed or exclusive in GBM cell lines have been identified. Among these, we found 11 specific potential targets for GBM including 5 mutated proteins such as RELL1, CYBA, EGFR, and MHC I proteins. Matching with drugs and clinical trials databases revealed that 7 proteins were druggable and under evaluation, 3 proteins have no known drug interaction yet and none of them are the mutated form of the identified proteins. Taken together, we discovered potential targets for immune therapy strategies in GBM.

IPLA2β Contributes to ER Stress-Induced Apoptosis during Myocardial Ischemia/Reperfusion Injury.

Both calcium-independent phospholipase A2 beta (iPLA2β) and endoplasmic reticulum (ER) stress regulate important pathophysiological processes including inflammation, calcium homeostasis and apoptosis. However, their roles in ischemic heart disease are poorly understood. Here, we show that the expression of iPLA2β is increased during myocardial ischemia/reperfusion (I/R) injury, concomitant with the induction of ER stress and the upregulation of cell death. We further show that the levels of iPLA2β in serum collected from acute myocardial infarction (AMI) patients and in samples collected from both in vivo and in vitro I/R injury models are significantly elevated. Further, iPLA2β knockout mice and siRNA mediated iPLA2β knockdown are employed to evaluate the ER stress and cell apoptosis during I/R injury. Additionally, cell surface protein biotinylation and immunofluorescence assays are used to trace and locate iPLA2β. Our data demonstrate the increase of iPLA2β augments ER stress and enhances cardiomyocyte apoptosis during I/R injury in vitro and in vivo. Inhibition of iPLA2β ameliorates ER stress and decreases cell death. Mechanistically, iPLA2β promotes ER stress and apoptosis by translocating to ER upon myocardial I/R injury. Together, our study suggests iPLA2β contributes to ER stress-induced apoptosis during myocardial I/R injury, which may serve as a potential therapeutic target against ischemic heart disease.

Novel Approach for The Electrophysiological Characterization of Mitochondrial Calcium Uniporter

Introduction The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types. Recently, we reported that the CCDC109A gene encodes a transcript variant shorter than the originally reported MCU (renamed “MCU-L”). Importantly, this short MCU variant (renamed “MCU-S”) lacks the mitochondrial target sequence at N-terminal but maintains its protein sequence identical to MCU-L. Hypothesis/Aim MCU-S variant can form functional Ca2+-permeable channels similar to MCU-L that are targeted to membranes other than the mitochondria, such as the plasma membrane (PM). Methods HEK293T cells were transfected with MCU variants and used for biochemical (cell surface protein biotinylation and protein fractionation), cell biological (confocal imaging of MCUs), and physiological assays (Ca2+ imaging and whole-cell patch-clamp). Results Overexpressed MCU-S was preferentially localized in the PM rather than in the mitochondria, while the MCU-L is exclusively expressed in the mitochondria. Using live-cell Ca2+ imaging, we demonstrated that overexpression of MCU-S induces larger Ca2+ influx across the PM compared to control cells, which is blocked by the conventional MCU blocker Ru360. Moreover, by whole-cell patch clamping, we found that cells overexpressing MCU-S exhibit an Ru360-sensitive inward current with a similar current-voltage relationship reported in MCU-L recording from mitoplasts. Lastly, co-transfection of MCU-S with proto-oncogene tyrosine-protein kinase (c-Src) and proline-rich tyrosine kinase 2 (Pyk2) significantly increased whole-cell MCU-S current compared to cells co-transfected with control vectors. Conclusion MCU-S forms a Ca2+ channel in the plasma membrane with similar characteristics of MCU-L. The physiological significance of this finding has yet to be determined, but our experiments suggest that it is possible to study MCU with a heterologous expression system, which uses considerably easier conditions compared to studying MCU and its regulation in the native inner mitochondrial membrane.

Acetylcholine Receptor Stimulation Mediates Trafficking of the Dopamine and Glutamate Transporters through Activation of Protein Kinase C

The dopamine transporter (DAT) clears neurotransmitter from the extracellular space and serves as an important regulator of signal amplitude and duration at sites of dopamine release. Similarly, the neuronal excitatory amino acid transporter 3 (EAAT3) plays an important role in the control of glutamatergic tone. Several different intracellular signaling pathways have been observed to modulate activity of these neurotransmitter transporters through regulation of the trafficking of the carriers to and from the cell surface. In this study, we investigated how the activation of protein kinase C (PKC) facilitates internalization of DAT and insertion of EAAT3 by addressing the endogenous cell-surface receptors that activate PKC and regulate the DAT and EAAT3 in vivo. We report here that stimulation of M1/M5 muscarinic receptors in midbrain cultures decreases the ability of dopamine neurons to transport dopamine through DAT. The cholinergic agonist, carbachol decreases DAT activity in primary cultures while the M1/M5-specific antagonist, pirenzepine, blocks these effects. The M3 antagonist, DAU 5884, has no effect, but a positive modulator of M5, VU 0238429, enhances the loss of DAT function in response to carbachol and acetylcholine. These data implicate M1/M5 receptors on dopamine neurons in the modulation of DAT function. Bisindolylmaleimide, a PKC inhibitor, blocks the effects of carbachol stimulation on dopamine uptake, indicating a role for PKC in muscarinic receptor-mediated DAT internalization. We designed a specific Gq inhibitor peptide that also blocks the effects of carbachol on DAT in primary cultures, confirming Gq as the G-protein that couples M1/M5 receptors to PKC activation in these cells. It has been established that the activity of the neuronal glutamate transporter EAAT3 increases in response to PMA treatment in the C6 glioma cell line. By total internal reflection fluorescence (TIRF) microscopy of a GFP-tagged EAAT3 we were able to confirm that this was due to an increase in EAAT3 present at the cell surface following PMA treatment. Carbachol activation of endogenous muscarinic receptors in the C6 glioma line also resulted in increased cell-surface expression of EAAT3. This increase was inhibited by co-application of the Gq inhibitor peptide indicating that the increase in EAAT3 activity depends upon Gq-coupled PKC signaling through M1/M5 receptors. In acute midbrain slices, biotinylation of cell-surface proteins showed that the decreases in dopamine transport mediated by muscarinic receptor stimulation the result of decreases in DAT carriers a cell surface. In addition, increases in surface expression of EAAT3 in the same experiment also appear to be modulated by M1/M5 receptors. These data indicate that stimulation of cholinergic receptors can lead to downregulation and upregulation of dopamine and glutamate transporters, respectively through alterations in their membrane trafficking.

Roles of Ezrin in regulation of ciliary beating in lung multiciliated cell

The mucociliary clearance is the first line of defense in the lungs. The function is given by the beating cilia on the surface of ciliated cells cooperating with the protective mucous layer. The beating cilia plays removing inhaled particles and pathogens (viruses, bacteria and fungi). Thus, the beating cilia are the key apparatus to conduct the mucociliary clearance. For ciliary beating to function properly, localization of ciliary proteins that regulate ciliary beating and formation of epithelial cell polarity are important. Ezrin/Radixin/Moesin (ERM) proteins function as general cross-linkers between apical membrane proteins and the actin cytoskeleton and are involved in the functional expression of apical membrane proteins. Especially, ezrin may play the important role of apical localization of β2-adrenergic receptor (β2AR) and cAMP-dependent protein kinase (PKA) in multiciliated cells. Here we examined roles of ezrin in the regulation of ciliary beating in lung multiciliated cells by using ezrin-knockdown (Vil2kd/kd) mice. To evaluate ciliary function, we analyzed ciliary beat frequency (CBF) and ciliary bend distance (CBD, and index of amplitude) in lung multiciliated cells of mice. The multiciliated cells were isolated by an elastase treatment. Ciliary beatings of lung multiciliated cells were observed by the videomicroscopy equipped with high speed camera (500 fps) and CBF and CBD were measured by an image analysis program using recorded images. We performed immunofluorescence microscopy analysis to evaluate the localization of β2AR, Na+/H+ exchanger regulatory factor (NHERF) 1, which is a PSD-95/Discs-large/ZO-1 (PDZ) scaffold protein, and PKA. In addition, we also performed cell surface protein biotinylation assay. In the results, stimulation with 1 nM procaterol, which is selective β2AR agonist, increased the ratios of CBF and CBD by about 80% in WT multiciliated cells. However, in Vil2kd/kd, stimulation with 1 nM procaterol increased the ratios of CBF and CBD by only 40%. While it is no significant difference that the increase of CBF and CBD by stimulation with 100 μM IBMX, which is a broad-spectrum phosphodiesterase inhibitor, in WT and Vil2kd/kd. In addition, the cell surface localization of NHERF1 and β2AR is disturbed in Vil2kd/kd multiciliated cells. There were no apparent changes in the subcellular localizations of PKA RIα and RIIα, which are PKA regulatory subunits, in Vil2kd/kd multiciliated cells. These results suggest that ezrin regulates the ciliary beating in lung multiciliated cells by promoting the apical localization of β2AR.

Acetylcholine Receptor Stimulation Activates Protein Kinase C Mediated Internalization of the Dopamine Transporter.

The dopamine transporter (DAT) clears neurotransmitters from the extracellular space and serves as an important regulator of signal amplitude and duration at sites of dopamine release. Several different intracellular signaling pathways have been observed to modulate DAT activity through the regulation of the trafficking of the carriers to and from the cell surface. Acute activation of protein kinase C (PKC) by phorbol esters facilitates clathrin-dependent internalization of the DAT in a variety of model systems; however, the physiological stimuli and cell-surface receptor systems that activate PKC and regulate the DAT in dopamine neurons remain elusive. We report here that stimulation of M1/M5 muscarinic receptors in midbrain cultures decreases the ability of dopamine neurons to transport dopamine through DAT. Application of the cholinomimetic drug carbachol leads to a decrease in DAT activity in primary cultures while the M1/M5-specific antagonist, pirenzepine, blocks these effects. The M3 antagonist, DAU 5884, does not affect, but a positive modulator of M5, VU 0238429, enhances the loss of DAT function in response to carbachol and acetylcholine. These data implicate M1/M5 receptors on dopamine neurons in the modulation of DAT function. Bisindolylmaleimide, a PKC inhibitor, blocks the effects of carbachol stimulation on dopamine uptake, supporting a role for PKC in muscarinic receptor-mediated DAT internalization. Furthermore, as shown previously for PKC-induced internalization, downregulation of the DAT is dependent on both clathrin and dynamin. A Gq-specific inhibitor peptide also blocks the effects of carbachol on DAT in primary cultures, confirming Gq as the G-protein that couples M1/M5 receptors to PKC activation in these cells. In acute midbrain slices, biotinylation of cell-surface proteins revealed the loss of dopamine transport mediated by muscarinic receptor stimulation was, indeed, due to loss of membrane expression of the DAT in endogenous tissue. These data indicate that stimulation of cholinergic pathways can lead to modulation of dopamine through internalization of the DAT.

USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.

The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R‐spondin induces auto‐ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R‐spondin by protecting ZNRF3/RNF43 from ubiquitin‐dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R‐spondin‐LGR4‐ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R‐spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.

Low Density Lipoprotein Receptor-Related Protein-1 (LRP1) Is Involved in the Uptake of Clostridioides difficile Toxin A and Serves as an Internalizing Receptor.

Toxin producing Clostridioides difficile strains cause gastrointestinal infections with the large glucosylating protein toxins A (TcdA) and B (TcdB) being major virulence factors responsible for the onset of symptoms. TcdA and TcdB enter their target cells via receptor-mediated endocytosis. Inside the cell, the toxins glucosylate and thereby inactivate small GTPases of the Rho-/Ras subfamilies resulting in actin reorganization and cell death. The receptors of TcdA are still elusive, glycoprotein 96 (gp96), the low density lipoprotein receptor family (LDLR) and sulfated glycosaminoglycans (sGAGs) have most recently been suggested as receptors for TcdA. In this study, we provide evidence on rapid endocytosis of Low density lipoprotein Receptor-related Protein-1 (LRP1) into fibroblasts and Caco-2 cells by exploiting biotinylation of cell surface proteins. In contrast, gp96 was not endocytosed either in the presence or absence of TcdA. The kinetics of internalization of TfR and LRP1 were comparable in the presence and the absence of TcdA, excluding that TcdA facilitates its internalization by triggering internalization of its receptors. Exploiting fibroblasts with a genetic deletion of LRP1, TcdA was about one order of magnitude less potent in LRP1-deficient cells as compared to the corresponding control cells. In contrast, TcdB exhibited a comparable potency in LRP1-proficient and -deficient fibroblasts. These findings suggested a role of LRP1 in the cellular uptake of TcdA but not of TcdB. Correspondingly, binding of TcdA to the cell surface of LRP1-deficient fibroblasts was reduced as compared with LRP1-proficient fibroblasts. Finally, TcdA bound to LRP1 ligand binding type repeat cluster II (amino acid 786–1,165) and cluster IV (amino acid 3332-3779). In conclusion, LRP1 appears to serve as an endocytic receptor and gp96 as a non-endocytic receptor for TcdA.

Tobacco Smoke and Inhaled Drugs Alter Expression and Activity of Multidrug Resistance-Associated Protein-1 (MRP1) in Human Distal Lung Epithelial Cells in vitro

Multidrug resistance-associated protein-1 (MRP1/ABCC1) is highly expressed in human lung tissues. Recent studies suggest that it significantly affects the pulmonary disposition of its substrates, both after pulmonary and systemic administration. To better understand the molecular mechanisms involved, we studied the expression, subcellular localization and activity of MRP1 in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and in the NCI-H441 cell line. Moreover, the effect of cigarette smoke extract (CSE) and a series of inhaled drugs on MRP1 abundance and activity was investigated in vitro. MRP1 expression levels were measured by q-PCR and immunoblot in AT2 and AT1-like cells from different donors and in several passages of the NCI-H441 cell line. The subcellular localization of the transporter was studied by confocal laser scanning microscopy and cell surface protein biotinylation. MRP1 activity was assessed by bidirectional transport and efflux experiments using the MRP1 substrate, 5(6)-carboxyfluorescein [CF; formed intracellularly from 5(6)-carboxyfluorescein-diacetate (CFDA)] in AT1-like and NCI-H441 cell monolayers. Furthermore, the effect of CSE as well as several bronchodilators and inhaled corticosteroids on MRP1 abundance and CF efflux was investigated. MRP1 protein abundance increased upon differentiation from AT2 to AT1-like phenotype, however, ABCC1 gene levels remained unchanged. MRP1 abundance in NCI-H441 cells were comparable to those found in AT1-like cells. The transporter was detected primarily in basolateral membranes of both cell types which was consistent with net basolateral efflux of CF. Likewise, bidirectional transport studies showed net apical-to-basolateral transport of CF which was sensitive to the MRP1 inhibitor MK-571. Budesonide, beclomethasone dipropionate, salbutamol sulfate, and CSE decreased CF efflux in a concentration-dependent manner. Interestingly, CSE increased MRP1 abundance, whereas budesonide, beclomethasone dipropionate, salbutamol sulfate did not have such effect. CSE and inhaled drugs can reduce MRP1 activity in vitro, which implies the transporter being a potential drug target in the treatment of chronic obstructive pulmonary disease (COPD). Moreover, MRP1 expression level, localization and activity were comparable in human AT1-like and NCI-H441 cells. Therefore, the cell line can be a useful alternative in vitro model to study MRP1 in distal lung epithelium.

Regulation of epidermal growth factor receptor expression and morphology of lung epithelial cells by interleukin-1β.

Accumulating evidences suggested that the overactivation of epidermal growth factor receptor (EGFR) was involved in the development of adult respiratory distress syndrome and pulmonary fibrosis. Elucidation of the mechanisms that regulate EGFR residence on the plasma membrane during inflammatory lung conditions is important for identifying potential therapies. We have demonstrated that flagellin phosphorylated EGFR at Ser1047 and induced transient EGFR internalization. In this study, we examined the molecular pathway and effect of interleukin 1 beta (IL-1β) on EGFR in alveolar epithelial cells. Treatment of A549 cells with IL-1β induced the activation of p38 mitogen-activated protein kinase (MAP kinase) and MAP kinase-activated protein kinase-2 (MAPKAPK-2), as well as EGFR phosphorylation at serine 1047. Both MAPKAPK-2 activation and EGFR phosphorylation were inhibited by SB203580, a p38 MAP kinase inhibitor. In addition, MK2a inhibitor (a MAPKAPK-2 inhibitor) suppressed EGFR phosphorylation. Assessment of the biotinylation of cell surface proteins indicated that IL-1β induced EGFR internalization. Furthermore, long-term treatment of A549 cells with IL-1β caused morphological changes and loss of cell-cell contact. Moreover, IL-1β augmented the effect of transforming growth factor beta 1 on the epithelial-mesenchymal transition. These results suggested that IL-1β regulates EGFR functions and induces morphological changes of alveolar epithelial cells.

The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of Its Cell Surface Expression and Viability of Tamoxifen-Resistant Breast Cancer Cells

Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

Limited AT1 Receptor Internalization Is a Novel Mechanism Underlying Sustained Vasoconstriction Induced by AT1 Receptor Autoantibody From Preeclampsia.

BackgroundAngiotensin II type 1 receptor (AT 1R) autoantibody (AT1‐AA) was first identified as a causative factor in preeclampsia. Unlike physiological ligand angiotensin II (Ang II), AT1‐AA can induce vasoconstriction in a sustained manner, causing a series of adverse effects, such as vascular injury and poor placental perfusion. However, its underlying mechanisms remain unclear. Here, from the perspective of AT 1R internalization, the present study investigated the molecular mechanism of sustained vasoconstriction induced by AT 1R autoantibody.Methods and ResultsIn the current study, we used the vascular‐ring technique to determine that AT1‐AA‐positive IgG, which was obtained from the sera of preeclamptic patients, induced long‐term vasoconstriction in endothelium‐intact or endothelium‐denuded rat thoracic arteries. The effect was caused by prolonged activation of AT 1R downstream signals in vascular smooth muscle cells, including Ca2+, protein kinase C, and extracellular signal‐regulated kinase 1 and 2. Then, using subcellular protein fractionation, cell surface protein biotinylation, and total internal reflection fluorescence, we found that AT1‐AA‐positive IgG resulted in significantly less AT 1R internalization than in the Ang II treatment group. Moreover, through use of fluorescent tracing and bioluminescence resonance energy transfer, we found that AT1‐AA‐positive IgG cannot induce the recruitment of β‐arrestin1/2, which mediated receptor internalization. Then, the effect of sustained AT 1R activation induced by AT1‐AA‐positive IgG was rescued by overexpression of β‐arrestin2.ConclusionsThese data suggested that limited AT 1R internalization resulting from the inhibition of β‐arrestin1/2 recruitment played an important role in sustained vasoconstriction induced by AT1‐AA‐positive IgG, which may set the stage for avoiding AT 1R overactivation in the management of preeclampsia.

PHB Regulates Trafficking of TRPM8 to the Plasma Membrane

TRPM8 is a Ca2+-permeable ion channel that mediates testosterone signaling in prostate epithelial cells. Previously, we demonstrated that TRPM8 is post-translationally modified by poly-R-3-hydroxybutyrate (PHB). Here, we tested whether removal of PHB from TRPM8 affects channel trafficking to the plasma membrane (PM). Using confocal microscopy, we found that PHB is highly colocalized with TRPM8 in the heterologous expression system and human prostate tissues. In cells transfected with PHB hydrolyzing enzyme PHB-depolymerase (PhaZ7) TRPM8 was no longer detected on the PM and showed an internalized pattern. The response of TRPM8 treated with PhaZ7 or PhaZ7-S136A, the mutant lacking enzymatic activity, to cold (15°C) or menthol demonstrated decreased TRPM8 activity with PhaZ7 treatment, but not the inactive form of the enzyme. Western Blot analysis of PhaZ7-treated TRPM8, following biotinylation of cell surface proteins, showed decreased TRPM8 expression on the PM upon PHB hydrolysis. Removal of PHB moieties from TRPM8 not only affected the channel trafficking to the PM but also directly impacted its function. As a result, testosterone-induced TRPM8 activity was completely abolished in planar lipid bilayers after the PhaZ7 treatment. In line with our earlier work, this inhibition was achieved from both sides of the channel but required more prolonged incubation at the inner side, further suggesting the prevalence of PHB-content on the internal side of the channel. Overall, these findings support the importance of PHBylation for TRPM8 trafficking to the PM and its function. Enzymatic cleavage of PHB could be responsible for downregulation of TRPM8 activity and its enhanced degradation in prostate cancer and present a possible mechanism of prostate cancer development.

Cell Surface Protein Biotinylation for SDS-PAGE Analysis.

Proteins expressed at the cell surface play important roles in physiology and represent valuable targets for new therapeutic agents. Indeed, the so-called druggable proteome consists, for about two thirds, of proteins that are either integral toor associated with the cell membrane. In spite of its importance, however, a complete characterization of the cell surface proteome has remained elusive because of the difficulty to efficiently purify these proteins from other contaminants. Methods exploiting the strong interaction between biotin and streptavidin have paved the way for the most significant advances in this field. The present chapter focuses on techniques for cell surface biotinylation with commercially available reagents and capture by avidin affinity chromatography and release of the biotinylated surface proteins for downstream analysis by electrophoretic methods.

Effects of salazosulfapyridine on the profile of cell surface proteins, revealed by biotinylation of cell surface proteins and 2-dimentional electrophoresis

ObjectiveWe investigated effects of salazosulfapyridine (SASP) on the protein profile of cell surface (CS)-proteins of SW982, a human synovial sarcoma cell line, using biotinylation of CS-proteins and 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE). MethodsSW982 cells were treated with SASP and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5ASA). Then the cells were treated with a membrane-impermeable biotinylating reagent. Biotinylated CS-proteins were isolated using NeutrAvidin-bound beads. CS-proteins affected by the drugs were detected by 2D-DIGE and subjected to mass spectrometry. ResultsBy the 2D-DIGE analysis, in total 576 spots were detected, 29 out of which showed more than ±1.5-fold different intensity in the SASP-, SP-, and 5ASA-treated cells, compared to non-treated cells (p < 0.05). Interestingly, 7 out of the 29 spots changed their intensity only by SASP and 17 spots changed their intensity only by SP. We identified 9 protein from 15 out of the 29 spots, most of which were evidenced to exist on the cell surface by flow cytometry. ConclusionWe found novel effects of SASP and its metabolites on SW982 cells by the combination of biotinylation of cell surface proteins and 2D-DIGE analysis. These data would help understanding of anti-rheumatic actions of SASP. Furthermore, the combination would be a useful method for the analysis of CS-proteins in various conditions.

Labeling Carboxyl Groups of Surface-Exposed Proteins Provides an Orthogonal Approach for Cell Surface Isolation.

Quantitative profiling of cell surface proteins is critically important for the understanding of cell-cell communication, signaling, tissue development, and homeostasis. Traditional proteomics methods are challenging for cell surface proteins due to their hydrophobic nature and low abundance, necessitating alternative methods to efficiently identify and quantify this protein group. Here we established carboxyl-reactive biotinylation for selective and efficient biotinylation and isolation of surface-exposed proteins of living cells. We assessed the efficiency of carboxyl-reactive biotinylation for plasma membrane proteins by comparing it with a well-established protocol, amine-reactive biotinylation, using SILAC (stable isotope labeling in cell culture). Our results show that carboxyl-reactive biotinylation of cell surface proteins is both more selective and more efficient than amine-reactive biotinylation. We conclude that it is a useful approach, which is partially orthogonal to amine-reactive biotinylation, allowing us to cast a wider net for a comprehensive profiling of cell surface proteins.